ARTICLE

Received 16 May 2014 | Accepted 30 Jun 2014 | Published 4 Aug 2014 DOI: 10.1038/ncomms5556

Gas regulates the post-endocytic sorting of -coupled receptors

Ste´phanie Rosciglione1, Caroline The´riault1, Marc-Olivier Boily1, Marile`ne Paquette1 & Christine Lavoie1

The role of Gas in G protein-coupled receptor (GPCR) signalling at the cell surface is well established. Recent evidence has revealed the presence of Gas on endosomes and its capacity to elicit GPCR-promoted signalling from this intracellular compartment. Here, we report an unconventional role for Gas in the endocytic sorting of GPCRs to lysosomes. Cellular depletion of Gas specifically delays the lysosomal degradation of GPCRs by disrupting the transfer of GPCRs into the intraluminal vesicles (ILVs) of multivesicular bodies. We show that

Gas interacts with GPCR-associated binding protein-1 (GASP1) and dysbindin, two key pro- teins that serve as linkers between GPCRs and the endosomal-sorting complex required for transport (ESCRT) machinery involved in receptor sorting into ILVs. Our findings reveal that

Gas plays a role in both GPCR signalling and trafficking pathways, providing another piece in the intertwining molecular network between these processes.

1 Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Universite´ de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4. Correspondence and requests for materials should be addressed to C.L. (email: [email protected]).

NATURE COMMUNICATIONS | 5:4556 | DOI: 10.1038/ncomms5556 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5556

eterotrimeric G proteins, which are composed of a, b Results and g subunits, transduce extracellular signals from Gas is required for the degradation of a subset of GPCRs.Gas HG protein-coupled receptors (GPCRs) to intracellular can localize to endosomes and participate in EGFR trafficking downstream effector proteins. In the conventional G protein- and signalling17–19. Because GPCRs are specifically sorted in signalling paradigm, the G protein localizes to the cytoplasmic endosomes, we investigated whether Gas is involved in the surface of the plasma membrane (PM), where after activation endocytic trafficking of these receptors. We first analysed the by an agonist-bound GPCR, the guanosine triphosphate (GTP)- impact of Gas depletion on the basal levels of various GPCRs that bound Ga and free Gbg bind to and regulate a number of are either sorted in the lysosomes (chemokine receptor type 4 well-studied effectors, including adenylyl cyclase, phospholipase (CXCR4), DOP, D2R and angiotensin 1 receptor (AT1R)) or Cb and ion channels1. However, over the past decade, research recycled to the PM (b2AR, m-opioid receptor (MOP) and has established that G proteins also have non-canonical roles in dopamine 1 receptor (D1R)) (Fig. 1a). Tagged GPCRs were 2–4 the cell, such as in regulating novel effectors , undergoing expressed in HEK293 cells transfected with control or Gas short GPCR-independent activation5 and acting on organelles2–6. interfering RNA (siRNA), and western blot (WB) analysis Indeed, in addition to localizing to the PM, heterotrimeric revealed that the expression levels of CXCR4, DOP, D2R and G proteins are found on the membranes of intracellular AT1R were increased in Gas-depleted cells, whereas the b2AR, compartments along both the endocytic and secretory MOP and D1R levels were unaffected. The quantification analysis pathways, where mounting evidence suggest they play several confirmed the higher levels of lysosome-targeted GPCRs in 5,7–10 roles in membrane trafficking . steady-state cells transfected with Gas siRNA (Fig. 1b), suggesting Longstanding evidence have suggested that Gas is involved in that the basal turnover of these receptors was delayed by Gas endosomal functions11–14. Several recent studies have confirmed knockdown. This result was confirmed with endogenous CXCR4. that Gas is present on endosomes and has non-conventional Gas depletion led to a significant increase in the steady-state roles in endosomal receptor signalling and trafficking15–19. The expression of endogenous CXCR4 in HeLa cells, a cell line ability of Gas to be activated and signal from this intracellular that expresses high levels of endogenous CXCR4 and Gas compartment was recently observed following the internalization (Supplementary Fig. 1a), suggesting that Gas similarly regulated of vasopressin type 2 receptor, parathyroid hormone receptor the basal turnover of an endogenous GPCR. The specificity of the (PTHR) and b2-adrenergic receptor (b2AR), all of which are Gas- effect of Gas on GPCR levels was validated in two different 15,16,20 coupled GPCRs .Gas also regulates the endosomal sorting manners. First, the depletion of Gai3 and Gb1 did not affect the and downregulation of epidermal growth factor receptor (EGFR), basal levels of haemagglutinin (HA)–CXCR4 or HA–DOP 18 a single transmembrane-spanning receptor . However, the role (Supplementary Fig. 1b). Second, after reintroducing Gas into of Gas in the endosomal sorting of other receptors, its precise Gas-depleted cells with siRNA-resistant forms of the short and molecular mechanism and the role of its activation status long variants of human Gas, the basal levels of HA–CXCR4 and (guanosine diphosphate (GDP)/GTP forms) in this trafficking HA–DOP were similar to or lower than those in the control cells step remain undefined. (Supplementary Fig. 1c), confirming a specific role for Gas in GPCR activity is tightly controlled by endocytic pathway. influencing the levels of these particular GPCRs. Interestingly, all Ligand-induced internalization drives GPCRs into early endo- of the GPCRs that were affected by Gas depletion are not coupled somes, where they are either recycled back to the PM for another to Gas in their conventional signalling pathways. Indeed, DOP, round of activation or sorted to the lysosomes for degradation, CXCR4 and D2R are coupled to Gai/o (refs 32–34), and AT1R is producing a prolonged attenuation of cellular signalling. coupled to Gai/q (ref. 35). Lysosomal sorting of GPCRs occurs via a highly conserved To determine whether the upregulated GPCRs in steady-state mechanism requiring recognition by the endosomal-sorting Gas-depleted cells localized to the PM, we measured the surface complex required for transport (ESCRT), which sorts receptors levels of these receptors as a function of Gas expression. We into the intraluminal vesicles (ILVs) of multivesicular bodies focused on CXCR4 and DOP because these two GPCRs were (MVBs), leading to a point of no return for complete degradation most affected by Gas depletion (Fig. 1a,b). A cell surface enzyme- in lysosomes21,22. Certain GPCRs are ubiquitinated, which linked immunosorbent assay (ELISA) demonstrated that the cell regulates their direct interaction with the hepatocyte growth surface expression of HA–DOP and HA–CXCR4 was signifi- factor regulated tyrosine kinase substrate (HRS) component of cantly increased in Gas-depleted HEK293 cells (Fig. 1c). As the the ESCRT machinery23–25. However, several GPCRs are sorted amount of a cell surface receptor is partly a function of the rate of by the ESCRT machinery independent of ubiquitination22,26,27. receptor internalization and recycling, we next examined the Recent work has demonstrated that GPCR-associated binding effect of Gas depletion on the internalization and recycling of protein-1 (GASP-1) and dysbindin link a subset of GPCRs to the HA-tagged CXCR4 and DOP by ELISA (Fig. 1d). No significant ESCRT machinery. GASP-1 binds to the carboxyl-termini of differences were observed between control and Gas-depleted cells 28–30 several GPCRs and targets them to the lysosomal pathway . (Fig. 1d), indicating that Gas depletion had no effect on the Dysbindin, a cytoplasmic protein that functions in the biogenesis internalization and recycling of these GPCRs and suggesting that of specialized lysosome-related organelles, has recently been Gas instead affected their constitutive trafficking and degradation. shown to promote the lysosomal sorting of d-opioid receptor Accordingly, immunofluorescence (IF) microscopy analysis of the (DOP) and dopamine 2 receptor (D2R) and is thought to link steady-state distribution of HA-tagged CXCR4 in HEK293 cells GASP-1 to the HRS component of the ESCRT machinery31. showed an accumulation of CXCR4 in intracellular vesicles in A mechanistic understanding of GPCR endosomal sorting is only Gas-depleted cells, whereas CXCR4 predominantly localized to beginning to emerge. the PM in control cells (Supplementary Fig. 1d). Together, these In the present study, we investigated whether Gas is involved in results suggested that the increased steady-state levels of CXCR4 the regulation of the endocytic sorting of GPCRs. Our results and DOP in Gas-depleted cells were owing to the involvement showed that Gas is critical for sorting GPCRs into the ILVs of of Gas in the constitutive trafficking and turnover of these MVBs through interactions with GASP-1, dysbindin and the receptors. ESCRT machinery. These interactions were independent of Gas We next investigated whether Gas depletion affected ligand- GTPase activity. This study defines a novel regulatory role for mediated GPCR trafficking and turnover. We assessed the effect Gas in the post-endocytic sorting and downregulation of GPCRs. of Gas depletion on the kinetics of the ligand-dependent

2 NATURE COMMUNICATIONS | 5:4556 | DOI: 10.1038/ncomms5556 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5556 ARTICLE

a HA- 3*HA- HA- Flag- HA- Flag- HA- b c CXCR4 DOP D2R AT1R β2AR MOP D1R 400 *** 200 **** Control siRNA + –+–+ –+–+–+–+– Control siRNA * 300 Gα siRNA 150 α s G s siRNA – + – + – + – + – + – + – + * 75 200 100 IB: ** *** HA/flag 100 50

50 GPCR relative Cell surface relative Cell surface expression (% control) expression 37 0 (% control) expression 0 α 50 IB: G s CXCR4 DOP IB: EEA1 150 DOP D2R AT1R β2AR MOP D1R CXCR4 f Control siRNA deHA - DOP 150 Gα siRNA Control siRNA s α Control siRNA Gα siRNA G s siRNA s * 100 * DPDPE(h): 0 23 4 0 23 4 HA-DOP HA-CXCR4 75 100 P=0.34 50 P=0.71 P=0.42 IB: HA 50 75 37 HA-DOP relative P=0.84 α T0) (% expression 50 IB:G s 37 0 IB:EEA1 150 0 1234 25 HA - CXCR4 Time (h) (% no treatment) 0 Control siRNA Gα siRNA 150 Cell surface expression Cell surface s ** α + No No α SDF1-α (h): 0 0.5 1 1.5 2 0 0.5 1 1.5 2 ** DPDPE SDF1 100 DPDPE + SDF1 Treatment Naloxone Treatment AMD3100 IB:HA 50 37 50 α IB:G s 37 T0) (% expression IB:EEA1 HA-CXCR4 relative 0 150 0.0 0.5 1.0 1.5 2.0 Time (h) g Flag-DOP HA-CXCR4 DPDPE 0 min 60 min 180 min SDF1α 0 min 60 min 120 min

Control Control siRNA siRNA

Gα s Gα siRNA s siRNA

Figure 1 | Gas knockdown delays GPCR degradation. (a) Representative immunoblots (IB) of the steady-state levels of various tagged GPCRs transiently expressed in HEK293 cells treated with control or Gas siRNA. EEA1, loading control. (b) Quantification of the GPCR expression in a. The data are presented as the percentage of GPCR expression compared with control cells. GPCRs in the left part of the histogram have a higher propensity to downregulate rather than to recycle and are not coupled to Gas for signalling. (c) Quantification of the cell-surface expression of HA–DOP and HA–CXCR4 in HEK293 cells treated with control or Gas siRNA. ELISAs were performed to measure cell-surface receptor expression. (d) Quantification of the internalization and recycling rates of HA–DOP and HA–CXCR4 in cells treated with control or Gas siRNA. ELISAs were performed to measure cell-surface receptor levels in HEK293 cells that were untreated, treated with agonist (5 mM DPDPE or 100 nM SDF1-a) for 30 min (to measure internalization) or treated with agonist followed by antagonist (10 mM naloxone or AMD3100) for 60 min (to measure recycling). Data are presented as the percentage of GPCR expression compared with untreated cells. (e) Representative immunoblots of the kinetics of Flag–DOP and HA–CXCR4 degradation following agonist stimulation in control or Gas-depleted HEK293 cells. Different film exposures were used for the control and Gas siRNA blots to better show the differences in the degradation kinetics. (f) Quantification of the GPCR expression in e. Data are presented as the percentage of GPCR expression compared with unstimulated cells (T0). (g) Immunofluorescence analysis of Flag–DOP and HA–CXCR4 internalization in HEK293 cells transfected with control or Gas siRNA. Cell-surface GPCRs were labelled with anti-Flag or anti-HA at 4 °C before incubation at 37 °C in the presence of agonists. At the indicated times, cells were processed for confocal microscopy analysis. For each time point, images were acquired using identical instrument settings. Scale bars, 10 mm. The data in b–d and f are presented as the mean±s.e.m. of Z3 independent experiments. Statistical analysis was performed using the Student’s t-test. *Pr0.05, **Pr0.01, ***Pr0.001, ****Pr0.0001. degradation of CXCR4 and DOP. Cells transiently overexpressing Gas siRNA, o20% of the receptors were degraded (Fig. 1f). HA-tagged CXCR4 or DOP and transfected with control or Gas The significantly reduced rate of receptor degradation in agonist- siRNA were treated with agonists in the presence of cyclohex- stimulated, Gas-depleted cells indicated a requirement for Gas in imide (to block de novo protein synthesis) for the indicated time, the establishment of ligand-mediated GPCR turnover, suggesting and GPCR abundance was monitored by WB and quantified that proper GPCR trafficking and sorting is dependent on Gas.IF (Fig. 1e). In cells transfected with control siRNA, 470% of the microscopy was utilized to evaluate the effects of Gas depletion DOP and CXCR4 receptors were degraded after stimulation on the endocytosis and degradation of cell-surface-labelled DOP for 4 h or 2 h, respectively, whereas in cells transfected with and CXCR4 (Fig. 1g). After agonist stimulation for 60 min, the

NATURE COMMUNICATIONS | 5:4556 | DOI: 10.1038/ncomms5556 | www.nature.com/naturecommunications 3 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5556 amount of DOP and CXCR4 that was internalized into vesicles endosome/lysosomes. In contrast, DOP and CXCR4 exhibited was similar in control and Gas-depleted cells, confirming that Gas robust colocalization with EEA1 in Gas siRNA-treated cells at this depletion did not impair GPCR internalization. However, after later time point. The quantitative analysis confirmed a significant longer agonist treatments (120 or 180 min), cell-surface-labelled increase in the colocalization of DOP and CXCR4 with EEA1- DOP and CXCR4 were mostly degraded in control cells, but positive endosomes in Gas-depleted cells compared with control remained in intracellular vesicles in Gas-depleted cells (Fig. 1g). cells (Fig. 2b). These results suggested that DOP and CXCR4 were Together, these results suggested that the decreased degradation trapped in early endosomes in the absence of Gas. of DOP and CXCR4 in Gas-depleted cells was due to retention in Following internalization in early endosomes, GPCRs destined intracellular compartments. for lysosomal degradation, such as DOP and CXCR4, are transferred from the endosome-limiting membranes to the ILVs of MVBs. We thus examined whether Gas altered the sorting of Gas promotes GPCR sorting in the ILVs of MVBs. To identify GPCRs into ILVs. DOP and CXCR4 were tagged at their C the intracellular compartment in which DOP and CXCR4 were terminus with green fluorescent protein (GFP) (DOP–GFP) or retained in Gas-depleted cells, we examined the distribution of cyan fluorescent protein (CFP) (CXCR4–CFP), and were co- internalized HA-tagged DOP and CXCR4 in control and Gas expressed with constitutively active Rab5 (Rab5-Q79L) to create siRNA-treated cells (Fig. 2a). After 15 min of agonist stimulation, enlarged endosomes and to facilitate the detection of these GPCRs cell-surface-labelled DOP and CXCR4 colocalized with the early on the limiting and intraluminal membranes of MVBs (Fig. 2c). endosomal marker EEA1 in both control and Gas-depleted cells. Confocal microscopy analysis of control cells stimulated with After 120 or 180 min of agonist stimulation, DOP and CXCR4 agonist for 90 min showed the presence of DOP–GFP and showed little colocalization with EEA1 in control cells, consistent CXCR4–CFP in both the limiting membranes and the ILVs of with receptor trafficking out of the early endosome to the late enlarged endosomes labelled with EEA1 (Fig. 2d). In contrast, in

a EEA1/Flag-DOP EEA1/HA-CXCR4 b DOP/EEA1 CXCR4/EEA1 ) 30 **** α 30 **** α 20 20

10 10 % Colocalization 0 % Colocalization 0 (120 min SDF1 (180 min DPDPE) 15 min SDF1- 15 min DPDPE α α Control G s Control G s siRNA siRNA siRNA siRNA c ILV α

GFP

GFP 180 min DPDPE 120 min SDF1-

α α Control siRNA G s siRNAControl siRNA G s siRNA Limiting membrane

Control siRNA α defG s siRNA DOP-GFP EEA1MergeDOP-GFP EEA1 Merge Control siRNA Control siRNA Gα siRNA α s G s siRNA 100 80 **** *** 90 min 75

DPDPE 60 40 60 50 40 CXCR4-CFPEEA1Merge CXCR4-CFP EEA1 Merge 25 20 α 0 Middle fluorescence 0 Normalized fluorescence 75 –25 0 25 50 100 125 (% of limiting membrane) 90 min SDF1 DOP-GFP CXCR4-CFP Normalized diameter

Figure 2 | Gas knockdown alters the transfer of GPCRs to the ILVs of MVBs. (a)Gas depletion prolonged the presence of internalized Flag–DOP and HA–CXCR4 in early endosomes. Confocal microscopy images comparing the distribution of the early endosome marker EEA1 and the cell-surface- labelled Flag–DOP and HA–CXCR4 in control or Gas-depleted HEK293 cells treated with agonist for 15, 120 or 180 min. Scale bars, 10 mm. The images were acquired at different laser and photomultiplier (PMT) settings to enable the detection of the GPCRs at each time point and to compare their localization with EEA1. (b) Quantification of the degree of overlap between EEA1 and HA–CXCR4 or Flag–DOP in cells treated with control or Gas siRNA. (c) Diagram illustrating the distribution of a GPCR with a C-terminal GFP tag on the outer membrane of an endosome and in ILVs. (d)Gas depletion prevented the redistribution of DOP–GFP and CXCR4–CFP from the endosome-limiting membrane to the ILVs. Representative optical sections depict endosomes in control and Gas-depleted HEK293 cells co-transfected with DOP–GFP or CXCR4–CFP along with Rab5-Q79L (to create enlarged endosomes) and treated with agonist for 90 min. Cells were fixed and processed for confocal microscopy. Scale bars, 10 mm. (e) Representative line scan analysis to quantify GPCR– GFP localization to the ILVs of endosomes. The normalized diameter represents the diameter of the endosome, where 0 and 100 correspond to the pixel distances with the first and second maximum pixel intensities, signifying the limiting membranes of the endosomes. The black and red traces represent the normalized fluorescence pixel intensity measured across the endosomes in control and Gas-depleted cells, respectively, with the maximum pixel intensity across the line normalized to 100. The hatched box highlights the normalized fluorescence values of pixels from 40 to 60% of the normalized diameter that were used to determine the mean intraluminal fluorescence for each endosome. (f) Compiled results of the line scan analysis for DOP–GFP and CXCR4–CFP. The data in b and f are presented as the mean±s.e.m. of nZ100 cells or endosomes, respectively, from three independent experiments. Statistical analysis was performed using the Student’s t-test. ***Po0.001, ****Pr0.0001.

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Gas-depleted cells, DOP–GFP and CXCR4–CFP localized pre- HRS-positive endosomes. Similarly, Cherry–GASP-1 was mainly dominantly to the limiting membranes of enlarged endosomes, distributed throughout the cytoplasm but also colocalized with with most endosomes exhibiting little intraluminal fluorescence Gas–GFP and endogenous HRS on sorting endosomes. (Fig. 2d). The GPCR distribution across the endosomes was Interestingly, the overexpression of Gas together with either quantified by line scan analysis of confocal cross-sections as dysbindin or GASP-1 altered the morphology of the early previously described36. A representative line scan analysis of an endosomes, as previously reported for overexpressed HRS38–40. endosome is shown in Fig. 2e. The peaks indicate the limiting Taken together, these data suggested that the components of this membrane, and the hatched box indicates the central region of the sorting complex localize together on early endosomes and could endosome lumen. An analysis of 4100 endosomes from multiple facilitate the sorting of GPCRs into the degradative pathway. In cells and experiments revealed a 50% reduction in both DOP–GFP agreement with this hypothesis, we confirmed that internalized and CXCR4–CFP in the endosomal lumen in Gas-depleted cells HA–DOP and HA–CXCR4 colocalized with Gas on early compared with control cells (Fig. 2f). Taken together, these results endosomes (Supplementary Fig. 3). indicated that Gas plays a crucial role in the sorting of GPCRs in the ILVs of MVBs for subsequent lysosomal degradation. Gas activation state does not alter GPCR endosomal sorting. To determine whether the effect of Gas on GPCR degradation depended on the GTPase activity of Gas, we investigated whether Gas is a component of the GPCR endosomal-sorting machinery. the interaction of Gas with the sorting components dysbindin, To decipher the molecular mechanism by which Gas regulates GASP-1 and HRS depended on the activation state of Gas. GPCR degradation, we next examined whether Gas associates Purified GST–Gas or GST alone preloaded with GDP (to mimic with the core machinery that mediates sorting into the ILVs of À the inactive state) or GDP/AlF4 or GTPgS (to mimic the active MVBs. ESCRT molecules, including HRS (ESCRT-0), are central 35 players in the endosomal sorting of GPCRs25,27. HRS can either state) was incubated with S-labelled, in vitro-translated GASP- 1, dysbindin or HRS, and protein binding was analysed (Fig. 4a). function directly by interacting with a ubiquitinated GPCR (such a as CXCR4)25 or indirectly by interacting with the accessory Inactive and active GST–G s bound dysbindin and GASP-1 at similar levels and did not bind HRS, suggesting that the Ga proteins GASP-1 and dysbindin, which are part of an alternate s connectivity network linking particular GPCRs (such as DOP and activation state did not influence these interactions. These results 28–31 were confirmed using HEK293 cell lysates overexpressing D2R) to the ESCRT machinery . To determine whether Gas associates with these endocytic sorting components, HEK293 cells Myc–dysbindin, Cherry–GASP-1 or untagged HRS (Fig. 4b). Again, the GST–Ga interactions with dysbindin, GASP-1 and were transiently transfected with GFP or Ga –GFP together with s s a untagged HRS, Myc-tagged dysbindin or Cherry-tagged GASP-1, HRS were independent of the activation state of G s. The small differences that were observed were not reproducible. The same and immunoprecipitations were performed with an anti-GFP results were obtained with immunoprecipitation assays using antibody. Gas–GFP interacted with HRS, dysbindin and GASP-1 41–43 Gas–GFP mutants mimicking active or inactive Gas (Fig. 3a), suggesting that Gas is part of this sorting machinery. (Supplementary Fig. 2b), indicating that the Gas activation state These interactions were specific for Gas, as the Ga proteins Gai3, Ga and Ga did not precipitate HRS, dysbindin or GASP-1 did not influence its interactions with GASP-1, dysbindin q z and HRS. (Supplementary Fig. 2a). a We next examined whether the interactions between Ga The signalling-independent role of G s in GPCR endosomal s sorting was next validated by determining whether the down- and the sorting machinery components are direct. 35S-labelled, a in vitro-translated GASP-1, dysbindin or HRS was incubated with stream effectors of G s altered CXCR4 downregulation. Because Ga stimulates cyclic AMP production and protein kinase A glutathione beads coated with glutathione S-transferase (GST) s alone or with the short and long forms of Ga fused to GST (PKA) activation, we tested whether forskolin, an adenylate s cyclase activator, could rescue Ga depletion and whether H89, a (GST–Ga S/L). As shown in Fig. 3b, GST–Ga bound to s s s a a dysbindin and GASP-1, but not HRS, suggesting that Ga bound PKA inhibitor, could mimic G s depletion. Control and G s- s depleted HEK293 cells expressing HA–CXCR4 were treated with directly to GASP-1 and dysbindin but required intermediate m m proteins to interact with HRS. Interestingly, dysbindin was vehicle (dimethylsulphoxide (DMSO)), 10 M forskolin or 10 M H89 for 8–12 h. CXCR4 abundance was monitored by WB identified by yeast two-hybrid as an interacting partner of HRS37, suggesting that dysbindin could be the link between Ga and (Fig. 4c) and quantified using scanning densitometry to compare s multiple independent experiments (Fig. 4d). PKA substrates were HRS. However, direct interactions between dysbindin and HRS or between dysbindin and GASP-1 have not been confirmed. To detected as a positive control for PKA activation (Fig. 4c). Although a significant increase in CXCR4 levels was observed in better define the complex containing GASP-1, dysbindin, HRS a and Ga , we generated GST–HRS and GST–GASP-1 and G s-depleted cells treated with DMSO, no significant differences s were observed between control cells (control siRNA) treated with examined their interaction with 35S-labelled, in vitro-translated Ga GASP-1, dysbindin or HRS (Fig. 3c). We confirmed that H89 and vehicle (DMSO) (Fig. 4d), indicating that PKA s, inactivation did not mimic Ga depletion. Furthermore, no dysbindin interacted directly with HRS, and that Ga interacted s s a directly with GASP-1 but not with HRS. GASP-1 also interacted significant differences were observed between G s siRNA-treated cells incubated with forskolin or vehicle (Fig. 4d), indicating that directly with HRS, but not with dysbindin. These results indicated a that, although dysbindin and GASP-1 do not interact, they PKA activation did not rescue the effect of G s knockdown on CXCR4 levels, which would be expected if the inhibition of cyclic provide links between Ga and HRS. s a We next investigated the intracellular localization of this AMP production in response to G s depletion was the cause of the increased CXCR4 levels. We concluded that Gas activity and protein complex. Gas has been shown to localize with HRS on early endosomes18. To determine whether dysbindin and GASP-1 its downstream effectors did not play a major role in GPCR endocytic sorting and downregulation. were present with Gas on HRS-labelled endosomes, we performed immunofluorescent confocal microscopy on COS7 cells co- expressing Gas–GFP and Myc–dysbindin or Cherry–GASP-1 Discussion (Fig. 3d). Myc–dysbindin had a largely diffuse cytoplasmic It is increasingly evident that endocytosis has numerous effects distribution, but a fraction colocalized with Gas–GFP on on GPCR signal transduction and that GPCR signalling regulates

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Myc – Cherry – a HRS Dysbindin GASP-1 GFP + – + – + – Gα - GFP – + – + – + IP: GFP s IB: HRS / Myc/GASP-1 100 50 250 75 75 lgG 75

50 50 IB: GFP 50 37 37 37

Lysates 25 25 25 IB: HRS / Myc/GASP-1 100 50 250

S/L α s b GST –+– c α GST GST-G GST-HRSGST-GASP-1 GST - G s S/L – – + 35 S HRS 100 35S HRS 100 35 S Dysbindin 50 35 S Dysbindin 50 250 35S GASP-1 250 50 35S GASP-1 35 α S G s Input Pull-down Input Pull-down d α G s-GFP Myc - HRS Merge Dysbindin

α Merge G s-GFP GASP-1 HRS

Figure 3 | Gas interacts and colocalizes with the GASP-1–dysbindin–HRS endosomal-sorting machinery. (a) Immunoprecipitation of Gas–GFP with HRS, Myc–dysbindin and Cherry–GASP-1. Lysates from HEK293 cells transiently transfected with GFP or Gas–GFP along with HRS, Myc–dysbindin or Cherry–GASP-1 were immunoprecipitated (IP) with anti-GFP and immunoblotted (IB) using the indicated antibody. (b) GST pull-down experiment 35 demonstrating that dysbindin and GASP-1, but not HRS, interact directly with Gas. In vitro translated S-labelled dysbindin and GASP-1 bound to GST–Gas 35 (short (S) and long (L) forms) but not to GST. S-labelled HRS did not bind to GST–Gas. Bound proteins were separated by SDS–PAGE and detected by autoradiography. (c) GST pull-down experiments to define the protein complex containing GASP-1, dysbindin, HRS and Gas. In vitro translated 35 S-labelled HRS, dysbindin, GASP-1 and Gas were incubated with GST alone, GST–Gas, GST–HRS or GST–GASP-1. The samples were analysed as in b.

(d) Immunofluorescence analysis of COS7 cells transfected with Gas–GFP and Myc–dysbindin or Cherry–GASP-1. The cells were fixed, labelled with anti-GFP, anti-HRS and anti-Myc or anti-Cherry and then processed for confocal microscopy. The overexpression of Gas together with dysbindin or GASP-1 promoted the formation of clustered and enlarged endosomes. Merged images illustrate the colocalization of Gas, endogenous HRS and dysbindin or GASP-1 on endosomes. Scale bars, 10 mm. the endocytic machinery. This has blurred the traditional lines level of GPCRs that are specifically targeted to lysosomes separating signalling and endocytosis at both the mechanistic and (CXCR4, DOP, D2R and AT1R). Previous studies have shown functional levels. Several proteins have been identified that that CXCR4 exhibits a high rate of constitutive internalization function in both signalling and endocytosis, the best example and turnover47,48, whereas other GPCRs, such as DOP, have a being the b-, which mediate GPCR endocytosis by slow rate of constitutive internalization and turnover49. These binding to AP2/clathrin and also participate in signal transduc- different turnover rates could explain why Gas knockdown had a tion by scaffolding components of the MAP-kinase pathway44–46. stronger effect on the basal levels of CXCR4. Further analysis of The present study determined that Gas, which is usually involved CXCR4 and DOP, which were most affected by Gas depletion, in GPCR signalling, is a cellular regulator of the post-endocytic indicated that Gas depletion significantly inhibited their sorting of lysosome-targeted GPCRs. lysosomal proteolysis following ligand-stimulated endocytosis This study indicated that Gas is involved in the regulation of without noticeably affecting their internalization and recycling both basal turnover and ligand-mediated degradation of GPCRs. rates. Furthermore, the reduced turnover of CXCR4 and DOP In steady-state cells, Gas depletion upregulated the expression was accompanied by their accumulation on the cell surface and in

6 NATURE COMMUNICATIONS | 5:4556 | DOI: 10.1038/ncomms5556 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5556 ARTICLE

Pull-down Input – γ a GDP GDP+AlF4 GTP S GST – + – + – + – + – α GST-G s S/L – – +– +–+–+

35S HRS 100

35S Dysbindin 50

35S GASP-1 250

Pull-down Lysates GDP GDP+AlF– GTPγS b 4 GST – + – + – + – + – α GST-G s S/L ––+ – + – + – +

IB: HRS 100

IB: Myc-Dysbindin 50 IB: Ch-GASP1 250

IB: Gβ1 37

cdHA-CXCR4 Control siRNA Gα siRNA DMSO FSK H-89 s 400 P = 0.11 Control siRNA + – + – + – α + + + P = 0.7 G s siRNA – – – 300 50 ** IB: HA 37 100 200 75 IB: PKA substrate 50 100 50 IB: Gα s 37 (% control siRNA - DMSO) 0 HA-CXCR4 relative expression IB: EEA1 150 DMSO FSK H89

Figure 4 | The interaction of Gas with the GPCR endosomal-sorting machinery is independent of the activation status of Gas. (a) In vitro GSTpull-down 35 experiment showing that the Gas activation state did not influence its direct interactions with HRS, dysbindin and GASP-1. In vitro translated S-labelled À HRS, dysbindin and GASP-1 were incubated with GST alone or GST–Gas preloaded with GDP (to mimic the inactive state) or GDP/AlF4 or GTPgS (to mimic the active state). Bound proteins were separated by SDS–PAGE and detected by autoradiography. (b) GST pull-down experiment in cell lysates demonstrating that the interactions between overexpressed dysbindin, GASP-1 and HRS were independent of the GST–Gas activation state. Lysates from HEK293 cells transiently transfected with HRS, Myc–dysbindin or Cherry–GASP-1 were incubated with GST alone, inactive GST–Gas–GDP or active À GST–Gas–GDP/AlF4 or GTPgS (as described in a). Bound proteins were analysed by immunoblotting (IB) for HRS, Myc (dysbindin) and Cherry (GASP-1). The interaction with Gb1 served as a control to confirm the activation state of GST–Gas because Gb1 only binds to inactive Gas. This control was included in each experiment in a,b.(c) The downstream effectors of Gas were not involved in CXCR4 downregulation. Control and Gas-depleted HEK293 cells expressing HA–CXCR4 were treated for 8 h with vehicle (DMSO), 10 mM forskolin (an adenylyl cyclase activator) or 10 mM H89 (a PKA inhibitor). The steady-state levels of HA–CXCR4 were analysed by immunoblotting using the indicated antibody. (d) Quantification of the HA–CXCR4 levels in c. The data are presented as the percentage of HA–CXCR4 compared with control siRNA cells treated with DMSO. The data are presented as the mean±s.e.m. of Z3 independent experiments and statistical analysis was performed using the Student’s t-test. **Pr0.01.

early endosomes, suggesting a role for Gas in the trafficking of endosomal-sorting components (Fig. 5). In this study, we these GPCRs through the sorting endosomes. The cellular determined that Gas interacts directly with GASP-1 and phenotype after Gas knockdown in this context closely dysbindin, two accessory sorting proteins that link a subset of resembled that observed when components of the ESCRT GPCRs (such as DOP and D2R) to the HRS component of the sorting machinery (such as HRS, AMSH and dysbindin) were ESCRT machinery in an ubiquitin-independent manner. Because 25,31,50 perturbed , implying that Gas is involved in the endosome- the interactions between GASP-1, dysbindin and HRS have been sorting pathway for GPCR trafficking to lysosomes. Indeed, previously reported but not clearly defined31,37, we further optical imaging demonstrated that Gas depletion reduced the characterized these interactions by showing that dysbindin endosomal sorting of DOP and CXCR4 into the ILVs of MVBs, interacts directly with HRS, but not with GASP-1, and that leading to the accumulation of DOP and CXCR4 on the limiting GASP-1 directly binds to HRS. Moreover, whereas human Gas membranes of early endosomes and preventing receptor was previously shown to directly interact with rat HRS18,no downregulation. interaction was detected between human Gas and human HRS, This work provides the first molecular insight into the suggesting that this interaction is species specific and is indirect in mechanism by which Gas regulates the lysosomal sorting of human cells. We propose that Gas is present on early endosomes, GPCRs. Our results indicated that Gas is required in both where direct interactions with GASP-1 and dysbindin promote ubiquitin-dependent and ubiquitin-independent HRS-mediated the downstream interaction of a subset of GPCRs with the ESCRT GPCR sorting, and suggested that Gas is a scaffold for machinery (Fig. 5a). These interactions enable the ubiquitin-

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Gas activity is clearly not limited to the cell surface. Evidence Recycling from multiple studies has indicated that Gas localizes to the endosomes, where it has a functional role in both receptor Early endosome signalling and trafficking. The presence of Gas on endosomes has 2AR D1R been known for more than a decade, but its role on this MOP intracellular compartment is only beginning to emerge. Gas is D2R now known to mediate functionally significant signalling from DOP endosomes. Various GPCRs (for example, thyroid stimulating a hormone receptor (TSHR), parathyroid hormone receptor, D1R and b2AR) signal via the Gas-linked activation of adenylyl cyclase directly from the endosome membrane15,20,56,57. Moreover, the GASP-1 HRS active forms of b2AR and Gas have been clearly visualized on

s Degradation

 endosomes15.Ga has also been implicated in endosomal G s Dysbindin membrane trafficking functions, such as endosome fusion, pIgR ESCRT 11–14,18,19 I,II,III transcytosis and EGFR downregulation . An important question is whether the GTPase activity of Gas is involved in endosomal trafficking. It was previously reported that inactive MVB CXCR4 Gas interacted with GIV/girdin and was involved in EEA1 membrane recruitment (for EGFR trafficking)19, and that the 14 active state of Gas negatively regulated endosomal fusion . b However, in our study, the Gas activation state did not affect the Ub HRS interactions with the endosomal-sorting machinery (GASP-1,

dysbindin and HRS). Consistent with these findings, the effect of s

α ? Gas was independent of the Gas-signalling effectors adenylyl G ESCRT cyclase and PKA, supporting an activation state-independent role I,II,III Lysosome for Gas. These results support a scaffolding, rather than a signalling role for Gas in GPCR degradation. Interestingly, we Figure 5 | Model for Gas regulation of GPCR endosomal sorting. Gas noted that none of the GPCRs affected by Gas depletion are promotes the endosomal sorting of GPCRs into the ILVs of MVBs via the coupled to Gas for signalling. Indeed, GPCRs coupled to Gas are ESCRT machinery. (a)Gas interacts directly with GASP-1 and dysbindin sorted to the PM following endocytosis. In fact, we were unable to on endosomal membranes and facilitates/stabilizes their downstream identify a GPCR coupled to Gas that is normally sorted to interactions with HRS and the ESCRT machinery. These interactions lysosomes. It is possible that the endosomal activation of Gas by promote the endosomal sorting and downregulation of a subset of GPCRs, these receptors prevents their interaction with the lysosomal- such as DOP and D2R, for which ESCRT sorting is ubiquitination sorting machinery, or perhaps the Gas-sorting step occurs later in independent. (b)Gas is also required for the endosomal sorting of GPCRs, endosomal maturation, when the recycling GPCRs have already such as CXCR4, that are sorted by the ubiquitin- and HRS-mediated ESCRT been removed. We intend to investigate these intriguing machinery, but are independent of GASP-1 and dysbindin. However, the hypotheses in future studies. The mechanism by which Gas is molecular components of the Gas-regulated CXCR4 endosomal-sorting translocated to endosomes remains unknown. One possibility is machinery have yet to be identified. that the activation of a Gas-linked GPCR stimulates translocation. Following b2AR activation or toxin treatment, Gas has been shown to dissociate from the PM through activation- 58–62 independent sorting of these GPCRs into the ILVs of MVBs, induced depalmitoylation of Gas . Another possibility is that resulting in lysosomal degradation. This model is supported by Gas is internalized through the endocytic pathway. Following the fact that Gas knockdown had similar effects on DOP and agonist stimulation, Gas has been shown to localize to vesicles D2R lysosomal sorting and degradation as the knockdown of derived from the PM that do not contain b2AR, suggesting that 31 27 63,64 GASP-1 (refs 30,51,52), dysbindin or HRS (Supplementary b2AR and Gas traffic through distinct endocytic pathways . Fig. 1b). The endosomal sorting role of Gas was not restricted to However, partial colocalization of Gas with b2AR has been GPCRs that interacted with GASP-1 and/or dysbindin. Indeed, observed on early endosomes15,63, which corresponds with our the proteolytic downregulation of CXCR4, which is sorted by the data indicating that Gas colocalizes with DOP or CXCR4 on ubiquitin- and HRS-mediated ESCRT machinery25 independent endosomes. Further studies are necessary to determine whether of GASP-1 (ref. 53) and dysbindin (Supplementary Fig. 1b), the stimulation of GPCRs that do not activate Gas, such as DOP, was also affected by Gas depletion. However, the molecular leads to the translocation of Gas to the endosomal membrane or components of the CXCR4 endosomal-sorting machinery that are whether the stimulation of a Gas-coupled GPCR increases the regulated by Gas have yet to be determined (Fig. 5b). Current translocation of Gas to endosomes, thereby modulating the studies are aimed at identifying this cofactor. Interestingly, the lysosomal degradation of other GPCRs. downregulation of EGFR, which is also sorted by the ubiquitin- Our findings raise the attractive possibility that Gas plays a role ESCRT machinery, has previously been shown to be altered by in both GPCR signalling and trafficking pathways, providing 18 Gas depletion , suggesting that Gas acts on a general component another piece to the intertwining molecular network between of the endosomal-sorting machinery for single and seven these processes. Thus, it is tempting to speculate that Gas plays a transmembrane receptors. Future studies should determine dual role in GPCR signalling via a rapidly responding second- whether Gas influences the downregulation of other GPCRs, messenger system and via the regulation of GPCR lysosomal such as protease-activated receptor 1 (PAR1), that are sorted to trafficking and downregulation. lysosomes independent of ubiquitination, GASP-1 and certain 26,54,55 late components of the ESCRT machinery . Future studies Methods should also refine our understanding of the role and significance Antibodies and reagents. The following antibodies were used in this study: of Gas in general endosomal sorting. anti-HA monoclonal antibody (mAb) (1:1,000 for WB and 1:500 for IF; Covance,

8 NATURE COMMUNICATIONS | 5:4556 | DOI: 10.1038/ncomms5556 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5556 ARTICLE

Emeryville, CA, USA), anti-Flag M1 and M2 mAbs and polyclonal antibodies Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, (pAbs) (1:1,000 for WB and 1:500 for IF; Sigma-Aldrich, Saint Louis, MO, USA), 10% glycerol and complete protease inhibitors) as previously described66. The anti-Gas pAb (1:1,000 for WB; Calbiochem, San Diego, CA, USA), anti-HRS mAbs lysates were incubated for 1 h at 4 °C and centrifuged at 15,000 g for 20 min at 4 °C. (1:100 for IF; Alexis Biochemicals, San Diego, CA, USA) and pAbs (1:1,000 for WB The supernatants were recovered, and the protein concentrations were evaluated by and 1:100 for IF; Millipore, Billerica, MA, USA), anti-GFP mAbs (1:3,000 for the Bradford assay. Thirty micrograms of each protein sample was aliquoted in WB and 1:500 for IF; Clontech, Mountain View, CA, USA) and pAbs (1:3,000 for Laemmli sample buffer and analysed by immunoblotting. For the degradation WB and 1:500 for IF; Invitrogen, Carlsbad, CA, USA), anti-EEA1 pAbs (1:1,000 assays, HEK cells were treated with the Gas siRNA duplex and transfected with for WB and 1:100 for IF; Thermo Scientific, Rockford, IL, USA), anti-EEA1 goat HA–CXCR4 or HA–DOP (as described above). Forty-eight hours after the initial Abs (1:100 for IF), anti-Gai3 and anti-Gb1 pAbs (1:1,000 for WB) (Santa Cruz siRNA treatment, the cells were passaged onto poly-L-lysine-coated six-well plates Biotechnology, Santa Cruz, CA, USA), anti-CXCR4 (CD184; 1:1,000 for WB; BD (Sigma-Aldrich) and grown for an additional 24 h. The cells were washed and Bioscience, Franklin Lakes, NJ, USA) and anti-Myc pAbs (1:2,000 for WB and incubated with DMEM containing 25 mM Hepes, 0.2% bovine serum albumin 1:1,000 for IF; Upstate, Temecula, CA, USA). The anti-dysbindin (1:500 for WB) (BSA) and 50 mgmlÀ 1 cycloheximide for 15 min at 37 °C. The cells were then and anti-GASP-1 (1:10,000 for WB and 1:2,000 for IF) pAbs were generous gifts incubated with the same medium supplemented with agonist (100 nM SDF1-a or from Dr Koh-Ichi Nagata (Institute for Developmental Research, Aichi Human 5 mM DPDPE) for various periods of time. The cells were washed with ice-cold Service Center, Japan) and Dr Fre´de´ric Simonin (Universite´ de Strasbourg, France), phosphate-buffered saline and lysed in RIPA buffer. The lysates were processed as respectively. described above and were analysed by immunoblotting.

DNA constructs. pCDNA3.1 vectors expressing either the long (L) or short (S) Cell-surface ELISA. Cell-surface ELISAs were performed as previously 67 forms of Gas were obtained from the Guthrie cDNA Resource Center (Missouri described . HEK cells were treated with the Gas siRNA duplex and transfected University of Science and Technology, Rolla, MO, USA). The Gas–GFP fusion with HA–CXCR4 or HA–DOP (as described above, 2 mg per P10 dish). Forty-eight protein was a generous gift from Dr Mark Rasenick (University of Illinois, Chicago, hours after the initial siRNA treatment, the cells were plated onto poly-L-lysine- 62 IL, USA) and has been previously described . The constitutively active Gas mutant coated 24-well plates (Sigma-Aldrich). After 24 h, the cells were starved for 1 h at (Q227L) and constitutively inactive Gas mutant (G226A, R280K, T284D, I285T 37 °C in DMEM and then incubated with DMEM containing 25 mM Hepes, 0.2% and A366S) were generated from Gas–GFP complementary DNA (cDNA) by BSA and agonist (100 nM SDF1-a or 5 mM DPDPE) for 30 min or agonist for QuickChange site-directed mutagenesis as previously described41–43. siRNA- 30 min followed by antagonist (10 mM Naloxone or AMD3100) for 60 min. The resistant forms of the WT, active and inactive Gas constructs were created by cells were then fixed with 3% formaldehyde, washed with Tris-buffered saline, introducing silent substitutions into the Gas or Gas–GFP cDNAs within the region blocked in 5% BSA and incubated for 1 h with primary antibody (monoclonal 18 of homology to the siRNA Gas oligo, as previously described . pcDNA3–Gai3– anti-HA antibody) and for 45 min with secondary antibody (alkaline phosphatase- 65 YFP has been previously described , and pcDNA3-Gaq and pcDNA3-Gaz were conjugated goat anti-mouse antibody; Sigma-Aldrich). The cells were then washed purchased from the Guthrie cDNA Resource Center (Missouri University of three times, and 250 ml of a colorimetric alkaline phosphatase substrate Science and Technology) and subcloned into pEGFP-N1. pRK5–Myc–dysbindin (diethanolamine and phosphatase substrate; Sigma-Aldrich) was added. The plates was obtained from Dr Koh-Ichi Nagata (Institute for Developmental Research, were incubated at 37 °C for the appropriate time, and then 250 ml of NaOH (0.4 M) Aichi Human Service Center, Japan), and untagged dysbindin was generated by was added to stop the reaction. A 100-ml aliquot of the colorimetric reaction was subcloning into pcDNA3 and pGEX-KG. pcDNA3–Cherry–GASP-1 and pGEX– collected, and the absorbance was measured at 405 nm using a spectrophotometer GASP-1 were obtained from Dr Fre´de´ric Simonin (Universite´ de Strasbourg, (Titertek Multiskan MCC/340; Labsystems). France), and untagged GASP-1 was obtained by subcloning into pcDNA3. pCS2–HRS–RFP was purchased from Addgene (Cambridge, MA, USA), and Immunoblotting untagged HRS was subcloned into pcDNA3 and pGEX-KG. pcDNA3–HA–CXCR4 . The protein samples were separated on 8 or 10% and pcDNA3–HA–b2AR were obtained from Dr Jean-Luc Parent (Universite´ de SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to nitro- Sherbrooke, QC, Canada). pcDNA3–Flag–AT1R and pEGFP–C1–DOP were cellulose membranes (Perkin Elmer, Waltham, MA, USA). The membranes were obtained from Dr Richard Leduc and Dr Louis Gendron (Universite´ de blocked in Tris-buffered saline (20 mM Tris–HCl, pH 7.4, and 150 mM NaCl) Sherbrooke, QC, Canada), respectively. pECFP–C1–CXCR4 was a generous gift containing 0.1% Tween 20 and 5% non-fat dry milk, incubated with primary ° from Dr Richard Miller (Northwestern University, Chicago, IL, USA). pcDNA3.1- antibodies for 1 h at room temperature or overnight at 4 C, subsequently incu- 3*HA–DOP, pcDNA3.1–HA–D1R and pcDNA3.1–HA–D2R were purchased from bated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG Missouri S&T, cDNA Resource Center (Missouri University of Science and (Bio-Rad, Hercules, CA, USA) and enhanced using a chemiluminescence detection Technology). reagent (Pierce Chemical, Thermo Fisher Scientific, Waltham, MA, USA).

Immunofluorescence Cell culture and transfection. COS7 cells were obtained from Dr Klaus Hahn . HEK or COS7 cells were grown on coverslips. Twenty- (University of North Carolina, NC, USA), and HEK293T cells were obtained from four to seventy-two hours after transfection, the cells were fixed with 3% Dr Alexandra Newton (University of California, San Diego, CA, USA). HEK293 paraformaldehyde in 100 mM phosphate buffer, pH 7.4, for 30 min, permeabilized cells stably expressing Flag–DOP or Flag–MOP were obtained from Dr Richard with 0.1% Triton X-100 for 10 min, blocked with 10% fetal bovine serum or goat Howells (New Jersey Medical School, Newark, NJ, USA). The cells were grown serum for 30 min, incubated with primary antibodies for 1 h at room temperature in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Invitrogen) and incubated with Alexa Fluor-conjugated secondary antibodies (Molecular containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), Probes, OR). The cells were visualized using an inverted confocal laser scanning penicillin and streptomycin. G418 (200 mgmlÀ 1, Invitrogen) was added to the microscope (FV1000; Olympus, Tokyo, Japan) equipped with a PlanApo 60x/1.42 culture medium for the stable cell lines. Cells were transfected using Lipofectamine oil immersion objective. Olympus FluoView version 1.6a was used to acquire and 2000 (Invitrogen), X-TremeGENE HP or Fugene 6 (Roche Diagnostic, analyse the images, which were further processed using Adobe Photoshop (Adobe Indianapolis, IN, USA) according to the manufacturers’ instructions. Systems, San Jose, CA, USA). The degree of colocalization between fluorescently labelled GPCRs and the early endosome marker EEA1 was quantified by calcu- lating the Manders coefficient68 using Olympus FluoView v1.6b colocalization RNA interference and rescue. Scrambled RNA oligos (scramble II duplex) and software. The quantitative analysis was performed on 30 size-matched cells for each 18 siRNAs against Gas (previously described in Zheng et al. ), Gai3,Gb1 and HRS experimental condition, and the experiments were performed twice. were purchased from Dharmacon (Lafayette, CO, USA), and dysbindin siRNA was purchased from Qiagen (Hilden, Germany). HEK293T cells were transfected with a Quantification of DOP–GFP localization in the endosomal lumen final concentration of 100 nM siRNA duplex using Lipofectamine 2000 . HEK cells a (Invitrogen) according to the manufacturer’s instructions. The cells were analysed treated with the control or G s siRNA duplex were transfected with DOP–GFP or 72 h after the siRNA transfection. The various tagged GPCR cDNAs were CXCR4–CFP along with Rab5-Q79L to create enlarged endosomes. Forty-eight transfected using X-tremeGENE HP 48 h before the cell lysis or IF experiments. hours later, the cells were plated onto poly-L-lysine-coated coverslips and incubated m a Rescue experiments were performed by transfecting the cells with cDNAs encoding in the presence of 5 M DPDPE or 100 nM SDF1- for 90 min before fixation, siRNA-resistant forms of untagged Ga (short and long) or with control vectors processing for IF and image acquisition. To quantify the presence of DOP–GFP s and CXCR4–CFP in the ILVs of endosomes, measurements were taken from raw (pcDNA3) using X-tremeGENE HP 10 h after the initial human Gas siRNA 36,69 transfection. data on individual endosomes as previously described . The quantification was performed on raw data representing confocal cross-sections of individual endosomes. For each endosome, straight-line selections were drawn across the GPCRs basal expression and degradation assay. For the basal expression diameter, and pixel intensities across the line were measured. The endosomal analysis, HEK293T cells were treated with the Gas siRNA duplex and transfected diameter was normalized to account for different endosome sizes. The pixel with the various tagged GPCRs (as described above). Seventy-two hours after the numbers with the first and second maximum pixel intensities, corresponding to initial siRNA transfection, all the cells were lysed in RIPA buffer (50 mM Tris-HCl, pixels on the limiting membrane of the endosome, were normalized to 0 and 100, pH 8, 150 mM NaCl, 1% NP40, 0.1% sodium deoxycholate, 5 mM EDTA and respectively. The location across the line of pixel 0 was then subtracted from each complete protease inhibitors (Roche, Basel, Switzerland)), with the exception of the pixel situated on the line, and this value was divided by the total diameter (in Flag–DOP and Flag–MOP-stable cell lines, which were lysed in OR buffer (150 mM pixels) of the endosome. This generated normalized pixel distances corresponding

NATURE COMMUNICATIONS | 5:4556 | DOI: 10.1038/ncomms5556 | www.nature.com/naturecommunications 9 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5556

to the distance across the line occupied by each pixel and was expressed as a 4. Dupre, D. J., Robitaille, M., Rebois, R. V. & Hebert, T. E. The role of percentage. The average background fluorescence was subtracted from the raw Gbetagamma subunits in the organization, assembly, and function of GPCR pixel intensity values. The pixel intensities for the pixel numbers normalized to signaling complexes. Annu. Rev. Pharmacol. Toxicol. 49, 31–56 (2009). 0 and 100 were also normalized to 0 and 100, respectively, generating normalized 5. Giannotta, M. et al. The KDEL receptor couples to Galphaq/11 to activate Src fluorescence values. The background-corrected pixel intensity values kinases and regulate transport through the Golgi. Embo J. 31, 2869–2881 corresponding to pixels that lay 40–60% across the endosomal diameter were (2012). averaged, generating a middle-fluorescence value for each endosome. The 6. Khan, S. M. et al. The expanding roles of Gbetagamma subunits in G middle-fluorescence values for multiple cells were compiled, and the mean for each protein-coupled receptor signaling and drug action. Pharmacol. Rev. 65, condition is shown. 545–577 (2013). 7. Irannejad, R. & Wedegaertner, P. B. Regulation of constitutive cargo transport from the trans-Golgi network to plasma membrane by Golgi-localized G Antibody uptake assay. HEK cells were treated with the control or Gas siRNA duplex and transfected with HA–CXCR4 or HA–DOP (as described above). protein betagamma subunits. J. Biol. Chem. 285, 32393–32404 (2010). Forty-eight hours after the initial siRNA treatment, the cells were passaged onto 8. Stow, J. L. & Heimann, K. Vesicle budding on Golgi membranes: regulation by poly-L-lysine-coated coverslips (BD Bioscience) and grown for an additional 24 h. G proteins and motors. Biochim. Biophys. Acta 1404, 161–171 (1998). After serum starvation for 1 h, the cells were incubated on ice for 1 h with DMEM 9. Nurnberg, B. & Ahnert-Hilger, G. Potential roles of heterotrimeric G proteins containing 25 mM Hepes, 0.2% BSA and anti-HA or anti-Flag (for stable cells) of the endomembrane system. FEBS Lett. 389, 61–65 (1996). antibody (1:500 dilution). The cells were washed in ice-cold DMEM containing 10. Bomsel, M. & Mostov, K. Role of heterotrimeric G proteins in membrane 25 mM Hepes and 0.2% BSA and were incubated at 37 °C with the same medium traffic. Mol. Biol. Cell 3, 1317–1328 (1992). supplemented with agonist (100 nM SDF1-a or 5 mM DPDPE) for different times. 11. Bomsel, M. & Mostov, K. E. Possible role of both the alpha and beta gamma Subsequently, the cells were fixed and processed for IF. To enable the direct subunits of the , Gs, in transcytosis of the polymeric comparison of the Flag–DOP or HA–CXCR4 levels remaining in the cells following immunoglobulin receptor. J. Biol. Chem. 268, 25824–25835 (1993). agonist treatment for different periods of time, all the images within a given 12. Beron, W., Colombo, M. I., Mayorga, L. S. & Stahl, P. D. In vitro reconstitution experiment were taken with the same magnification and laser intensity settings. of phagosome-endosome fusion: evidence for regulation by heterotrimeric . Arch. Biochem. Biophys. 317, 337–342 (1995). 13. Colombo, M. I., Mayorga, L. S., Casey, P. J. & Stahl, P. D. Evidence of a role Co-immunoprecipitation. HEK cells were transiently transfected with GFP or for heterotrimeric GTP-binding proteins in endosome fusion. Science 255, Gas–GFP together with HRS, Myc–dysbindin or Cherry–GASP-1 cDNA. 1695–1697 (1992). Forty-eight hours later, the cells were washed twice with ice-cold phosphate- 14. Colombo, M. I., Mayorga, L. S., Nishimoto, I., Ross, E. M. & Stahl, P. D. Gs buffered saline, lysed in 50 mM Tris–HCl pH 7.4, containing 150 mM NaCl, 1% regulation of endosome fusion suggests a role for signal transduction pathways NP40 and protease inhibitors for 1 h at 4 °C and centrifuged at 15,000 g for 20 min. in endocytosis. J. Biol. Chem. 269, 14919–14923 (1994). The cleared supernatants were incubated with primary antibodies (1 mg of antibody 15. Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from ° per 1 mg of protein) overnight at 4 C and then with protein A-Sepharose (GE endosomes. Nature 495, 534–538 (2013). Healthcare, Piscataway, NJ, USA) or protein G-Sepharose (Zymed, San Francisco, 16. Feinstein, T. N. et al. Noncanonical control of vasopressin receptor type 2 CA, USA) beads for 1 h. The beads were washed three times in lysis buffer and then signaling by retromer and . J. Biol. Chem. 288, 27849–27860 (2013). boiled in Laemmli sample buffer. Bound immune complexes were analysed by 17. Van Dyke, R. W. Heterotrimeric G protein subunits are located on rat liver SDS–PAGE and immunoblotting. endosomes. BMC Physiol. 4, 1 (2004). 18. Zheng, B. et al. Regulation of epidermal growth factor receptor degradation by GST pull-down assays. GST-fusion proteins were expressed in Escherichia coli heterotrimeric Galphas protein. Mol. Biol. Cell 15, 5538–5550 (2004). BL21 cells and purified on glutathione-Sepharose 4B beads (Pharmacia, Piscataway, 19. Beas, A. O. et al. Galphas promotes EEA1 endosome maturation and shuts NJ, USA) as previously described70.The35S-labelled in vitro translation products down proliferative signaling through interaction with GIV (Girdin). Mol. Biol. of pcDNA3–dysbindin, pcDNA3–GASP-1 and pcDNA3–HRS were prepared using Cell 23, 4623–4634 (2012). the TNT T7 rabbit reticulocyte Quick Coupled Transcription/Translation system 20. Ferrandon, S. et al. Sustained cyclic AMP production by parathyroid hormone (Promega, San Luis Obispo, CA, USA) in the presence of [35S]EasyTag EXPRESS receptor endocytosis. Nat. Chem. Biol. 5, 734–742 (2009). labelling mix (73% Met/22% Cys; 41,000 Ci/mmol, Perkin Elmer). A total of 21. Wollert, T. & Hurley, J. H. Molecular mechanism of multivesicular body 5–10 mg of purified GST or GST-fusion protein was incubated with the in vitro biogenesis by ESCRT complexes. Nature 464, 864–869 (2010). translated products in 20 mM Tris, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.1% 22. Hanyaloglu, A. C. & von Zastrow, M. Regulation of GPCRs by endocytic NP40, 1 mM dithiothreitol and complete protease inhibitor for 2 h at 4 °C and membrane trafficking and its potential implications. Annu. Rev. Pharmacol. washed four times with the same buffer. Bound proteins were eluted with Laemmli Toxicol. 48, 537–568 (2008). buffer, resolved by SDS–PAGE and visualized by autoradiography. For the GST 23. Giordano, F., Simoes, S. & Raposo, G. The ocular albinism type 1 (OA1) GPCR pull-down assays on cell lysates, purified GST-proteins were incubated with 1 mg is ubiquitinated and its traffic requires endosomal sorting complex responsible of lysate from HEK cells expressing Myc–dysbindin, Cherry–GASP-1, untagged for transport (ESCRT) function. Proc. Natl Acad. Sci. USA 108, 11906–11911 HRS or Gas (S/L) that was prepared as described in the co-immunoprecipitation (2011). section. The bound proteins were separated by SDS–PAGE and detected by 24. Hasdemir, B., Bunnett, N. W. & Cottrell, G. S. Hepatocyte growth factor- immunoblotting. In the experiments involving nucleotide loading, GST–Gas was regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of preincubated with 30 mM GDP alone, 30 mM GDP, 30 mM AlCl3 and 10 mM NaF protease-activated receptor 2 and calcitonin receptor-like receptor. J. Biol. 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Recycling and (Northwestern University), Richard Leduc, Jean-Luc Parent and Louis Gendron resensitization of delta opioid receptors. DNA Cell Biol. 19, 195–204 (2000). (Universite´ de Sherbrooke) for generous gifts of antibodies, cDNA and cell lines. We also 50. Sierra, M. I., Wright, M. H. & Nash, P. D. AMSH interacts with ESCRT-0 to thank Jade Degrandmaison for the rapid development of ELISAs and Jean-Luc Parent regulate the stability and trafficking of CXCR4. J. Biol. Chem. 285, 13990–14004 for constructive comments on the manuscript. This work was supported by grant (2010). MOP-97936 from the Canadian Institutes of Health Research and a Canada Research 51. Cho, D. I. et al. ARF6 and GASP-1 are post-endocytic sorting proteins Chair in Cellular Pharmacology (to C.L.). selectively involved in the intracellular trafficking of dopamine D(2) receptors mediated by GRK and PKC in transfected cells. Br. J. Pharmacol. 168, Author contributions 1355–1374 (2013). 52. 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56. Kotowski, S. J., Hopf, F. W., Seif, T., Bonci, A. & von Zastrow, M. Endocytosis How to cite this article: Rosciglione, S. et al. Gas regulates the post-endocytic sorting of promotes rapid dopaminergic signaling. Neuron 71, 278–290 (2011). G protein-coupled receptors. Nat. Commun. 5:4556 doi: 10.1038/ncomms5556 (2014).

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